Abstract
BackgroundOleaginous microalgae represent a valuable resource for the production of high-value molecules. Considering the importance of omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs) for human health and nutrition the yields of high-value eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) require significant improvement to meet demand; however, the current cost of production remains high. A promising approach is to metabolically engineer strains with enhanced levels of triacylglycerols (TAGs) enriched in EPA and DHA.ResultsRecently, we have engineered the marine diatom Phaeodactylum tricornutum to accumulate enhanced levels of DHA in TAG. To further improve the incorporation of omega-3 LC-PUFAs in TAG, we focused our effort on the identification of a type 2 acyl-CoA:diacylglycerol acyltransferase (DGAT) capable of improving lipid production and the incorporation of DHA in TAG. DGAT is a key enzyme in lipid synthesis. Following a diatom based in vivo screen of candidate DGATs, a native P. tricornutum DGAT2B was taken forward for detailed characterisation. Overexpression of the endogenous P. tricornutum DGAT2B was confirmed by qRT-PCR and the transgenic strain grew successfully in comparison to wildtype. PtDGAT2B has broad substrate specificity with preferences for C16 and LC-PUFAs acyl groups. Moreover, the overexpression of an endogenous DGAT2B resulted in higher lipid yields and enhanced levels of DHA in TAG. Furthermore, a combined overexpression of the endogenous DGAT2B and ectopic expression of a Δ5-elongase showed how iterative metabolic engineering can be used to increase DHA and TAG content, irrespective of nitrogen treatment.ConclusionThis study provides further insight into lipid metabolism in P. tricornutum and suggests a metabolic engineering approach for the efficient production of EPA and DHA in microalgae.
Highlights
Oleaginous microalgae represent a valuable resource for the production of high-value molecules
In the work presented here, we studied the effect of transgenic expression of the three most promising candidate DGAT2s, namely P. tricornutum DGAT2A, DGAT2B and T. pseudonana DGAT2 (TpDGAT2), on lipid accumulation and omega-3 Long-chain polyunsaturated fatty acid (LC-PUFA) incorporation into TAG in wild type (WT) and transgenic strains
In P. tricornutum during N-replete and N-deplete conditions, TAGs and the precursor DAG are mainly composed of 16:0 and 16:1, whereas eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are minor species [28, 31]. This suggests that LC-PUFAs are likely transferred to TAGs by either one of the four DGAT2s identified in P. tricornutum [13] via the Kennedy pathway or a route involving phosphatidyl diacylglycerol acyltransferase Phospholipid diacylglycerol acyltransferase (PDAT) [2]
Summary
Oleaginous microalgae represent a valuable resource for the production of high-value molecules. Often microalgae accumulate high quantities of TAGs in response to abiotic stress, e.g. nutrient starvation, high temperature, salinity, pH or light intensity [4]. The application of such environmental stresses can be an effective strategy for increasing lipid production in microalgae; their negative effect on cell growth is a major bottleneck for reducing production costs. A promising approach is the use of genetic engineering for generating improved strains with desirable characteristics such as fatty acid (FA) composition, enhanced lipid biosynthesis and high growth rates
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